Hydrogen bond surrogate macrocycles as modulators of Ras

ABSTRACT

The present invention relates to peptides having one or more stable, internally-constrained HBS α-helices, where the peptide is capable of interacting with Ras and related proteins.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a national stage application under 35 U.S.C. 371 ofPCT Application No. PCT/US2012/027617, filed Mar. 2, 2012, which claimsthe benefit of U.S. Provisional Patent Application Ser. No. 61/449,472,entitled “Hydrogen Bond Surrogate Macrocycles as Modulators of Ras,”filed on Mar. 4, 2011, which is incorporated herein by reference in itsentirety.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with government support under grant numbersR01GM073943 and R01GM078266, both awarded by the National Institutes ofHealth. The government has certain rights in this invention.

BACKGROUND OF THE INVENTION

Aberrant receptor tyrosine kinase (RTK) signaling is a major underlyingcause of various developmental disorders and hyperproliferative diseases(Blume-Jensen et al., “Oncogenic kinase signalling”. Nature 2001, 411,355). A primary transduction mechanism by which RTK signals arepropagated to intracellular pathways involves the ligand-dependentactivation of the small guanine nucleotide binding protein Ras (FIG. 1)(Buday et al., “Many faces of Ras activation.” Biochim. Biophys. Acta2008, 1786, 178). Accordingly, design of Ras signaling pathwayinhibitors has been an active area of research for anticancer therapy(Downward et al., “Targeting Ras signalling pathways in cancer therapy.”Nat. Rev. Cancer 2003, 3, 11). The rate-limiting step in Ras activationprocess is the conversion of Ras-GDP to Ras-GTP through an exchangereaction that is catalyzed by the Ras specific guanine nucleotideexchange factor Sos (FIG. 2). The highly conserved catalytic domain(Rem+cdc25) of Sos interacts with Ras at a helical hairpin composed ofthe α-H and α-I helices (FIG. 3). The helical hairpin may be capable ofnucleotide dissociation from Ras and subsequent down-regulation of theRas pathway (Sacco et al., “The isolated catalytic hairpin of theRas-specific guanine nucleotide exchange factor Cdc25(Mm) retainsnucleotide dissociation activity but has impaired nucleotide exchangeactivity.” Febs Lett. 2005, 579, 6851). The high resolution structuresof this complex suggest that the α-H helix is the only portion of thehelical hairpin that makes direct contact with Ras, while the α-I helixmay only serve to stabilize the α-H conformation (Boriack-Sjodin, et al.“The structural basis of the activation of Ras by Sos.” Nature 1998,394, 337).

Inhibitors of the Ras-Sos interactions would be valuable as tools todissect this complex signaling pathway and as leads for anticancer drugdesign. However, despite the availability of a high resolution crystalstructure of the Ras/Sos complex since 1998, direct inhibitors of thiscomplex have not been reported. Therefore, there remains a need formethods and compositions for treating developmental disorders andhyperproliferative diseases by inhibiting undesirable activitiesassociated with Ras proteins, for example by inhibition of the Ras/Soscomplex. The invention addresses these and other needs.

SUMMARY OF THE INVENTION

In one aspect, the invention provides a peptide having a stable,internally-constrained alpha-helix, wherein said alpha helix isconstrained by a crosslink formed by a carbon-carbon bond-formingreaction, and further wherein the peptide mimics at least a portion of aprotein capable of interacting with a Ras protein. In some embodiments,the protein capable of interacting with a Ras protein is Sos. Forexample, the peptide mimics at least a portion of an αA, αB, αC, αD, αE,αF, αG, αH, αI, αJ, or αK helix of Sos. In some instances, the peptidemimics at least a portion of the a-H helix of Sos. In one embodiment,the peptide mimics amino acids 929-944 of the α-H sequence of Sos. Forexample, the peptide comprises a sequence of the formulaFXGZZXZXZLXZEXXN (SEQ ID NO: 1) where X is any amino acid residue and Zis a hydrophobic residue. In other embodiments, the peptide comprises anamino acid sequence of Table 1, and has an internally-constrainedalpha-helix spanning residues 1 through 4 of the amino acid sequence.

In some embodiments, the peptide comprises the formula:

where

-   is a single or double carbon-carbon bond;-   is a single bond and is cis or trans when    is a double bond;-   n is 1 or 2;-   m is zero or any positive integer;-   R₁, R₂, R₃ and R₄ are independently hydrogen, an amino acid side    chain, an alkyl group, or an aryl group.

For example, m is 1 or 2. In other embodiments, n is 1 or 2.

The invention also provides a pharmaceutical composition a peptideaccording to the invention and a pharmaceutically acceptable vehicle.

In another aspect, the invention provides a method of inhibiting Rassignaling in a cell, comprising contacting the cell with an effectiveamount of a composition comprising a peptide having a stable,internally-constrained alpha-helix, wherein said alpha helix isconstrained by a crosslink formed by a carbon-carbon bond-formingreaction, and further wherein the peptide mimics at least a portion of aprotein capable of interacting with a Ras protein. In some embodiments,the protein capable of interacting with a Ras protein is Sos. Forexample, the peptide mimics at least a portion of an αA, αB, αC, αD, αE,αF, αG, αH, αI, αJ, or αK helix of Sos. In some instances, the peptidemimics at least a portion of the α-H helix of Sos. In one embodiment,the peptide mimics amino acids 929-944 of the α-H sequence of Sos. Forexample, the peptide comprises a sequence of the formulaFXGZZXZXZLXZEXXN (SEQ ID NO: 1) where X is any amino acid residue and Zis a hydrophobic residue. In other embodiments, the peptide comprises anamino acid sequence of Table 1, and has an internally-constrainedalpha-helix spanning residues 1 through 4 of the amino acid sequence.

In some embodiments, the peptide comprises the formula:

where

-   is a single or double carbon-carbon bond;-   is a single bond and is cis or trans when    is a double bond;-   n is 1 or 2;-   m is zero or any positive integer;-   R₁, R₂, R₃ and R₄ are independently hydrogen, an amino acid side    chain, an alkyl group, or an aryl group.

For example, m is 1 or 2. In other embodiments, n is 1 or 2.

The invention further provides a method of treating cancer in a subjectin need thereof, comprising administering to the subject a peptidehaving a stable, internally-constrained alpha-helix, wherein said alphahelix is constrained by a crosslink formed by a carbon-carbonbond-forming reaction, and further wherein the peptide mimics at least aportion of a protein capable of interacting with a Ras protein. Forexample, the peptide comprises a sequence of the formulaFXGZZXZXZLXZEXXN (SEQ ID NO: 1) where X is any amino acid residue and Zis a hydrophobic residue. In other embodiments, the peptide comprises anamino acid sequence of Table 1, and has an internally-constrainedalpha-helix spanning residues 1 through 4 of the amino acid sequence.Insome embodiments, the peptide comprises the formula:

where

-   is a single or double carbon-carbon bond;-   is a single bond and is cis or trans when    is a double bond;-   n is 1 or 2;-   m is zero or any positive integer;-   R₁, R₂, R₃ and R₄ are independently hydrogen, an amino acid side    chain, an alkyl group, or an aryl group.

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 shows a schematic of the Ras signalling pathway.

FIG. 2 shows the relationship between Ras and Sos. The activity of Rasis facilitated by the specific guanine nucleotide exchange factor Sos.Activated Ras controls a multitude of signaling transduction pathways.

FIG. 3 shows the key α-helical interface between Ras and Sos (PDBaccession number 1BKD).

FIG. 4 shows inhibition of Sos-mediated guanine nucleotide exchangeactivity by compositions of the invention.

FIG. 5A shows selected changes in ¹H—¹⁵N HSQC spectra of ¹⁵N-labelledRas resonances upon addition of three and five equivalents of HBS 7.

FIG. 5B shows a plot of mean chemical shift differences illustratingchanges upon the addition of increasing amounts of HBS 7.

FIG. 6 shows inhibition of the Ras/Sos complex by HBS 7 as evaluated ina GST pull-down assay in the presence of 40 nM Sos and 100 nM GST-Ras.

FIG. 7 shows a schematic of the RTK/Ras/MAPK signaling network leadingto gene expression.

FIGS. 8 a-d show the effect of HBS peptides of the invention uponRTK/Ras pathway proteins as determined by experiments on cell cultures.FIG. 8 a shows attenuation of EGF-induced Ras activation by HBS 7. FIG.8 b shows downregulation of Ras activation by direct interference withthe Ras/Sos complex. FIG. 8 c shows suppression of EGF-induced ERKactivation by HBS 7. FIG. 8 d shows a reduction in the intensity andduration of EGF-induced ERK activation following treatment with HBS 7.

FIG. 9 shows circular dichroism spectra and helicity of HBS peptides.

FIG. 10 shows the binding affinity of HBS peptides for Ras as determinedby a fluorescence polarization assay.

FIG. 11 shows cellular uptake of HBS peptides into live HeLa cells.

FIG. 12 shows a synthetic scheme for preparation of HBS 13 and relatedcompounds.

FIG. 13 shows a synthetic scheme for preparation of HBS 7 and relatedcompounds.

FIG. 14 shows a synthetic scheme for preparation of various HBS peptidesof the invention.

FIG. 15 shows a synthetic scheme for the preparation of HBS peptidescontaining a glycine residue at the third position from the N-terminus.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to hydrogen bond surrogate (“HBS”)-derivedα-helices capable of disrupting the Ras signaling pathway. These HBShelices can potentially function as in vivo inhibitors of Ras/Sosinteraction.

A first aspect of the present invention relates to a peptide having oneor more stable, internally-constrained HBS α-helices, where the peptidemimics at least a portion of a protein capable of interacting with a Rasprotein. For example, the peptide mimics an alpha-helical portion of theprotein capable of interacting with a Ras protein.

The term “mimic” refers to the ability of a composition of the inventionto effect a similar activity as a natural protein such as Sos. A “mimic”encompasses both functional and structural mimics of such proteins. Forexample, the mimic is a protein which shares a certain percent homology(e.g. 60%, 70%, 80%, 85%, 90%, or 95% homology) with the target protein.Alternatively, the mimic is derived from a different sequence thatnevertheless is capable of interacting with Ras in a functionallysimilar manner, for example by interacting with the same active site.

Peptides according to the invention mimic, for instance, a portion ofthe sequence of a guanine-nucleotide-exchange factor such as the Sosprotein. Other nucleotide exchange factors include Cdc25, Sdc25 andRasGRF. The Sos protein comprises two alpha-helical structural domains,including an N-terminal domain (amino acids 568-741, encompassingα-helices α1 through α6) and a C-terminal domain (amino acids 752-1044,encompassing α-helices αA-αK). The C-terminal domain of the Sos proteinis involved primarily in interaction with Ras. In particular, helix αHplays an important role in the nucleotide exchange mechanism.

In some embodiments, suitable peptides of the invention mimic at leastone α-helix which is an αA, αB, αC, αD, αE, αF, αG, αH, αI, αJ, or αKhelix of Sos. For instance, peptides mimic the αH or αI helix of Sos. Insome embodiments, peptides of the invention mimic amino acids 929through 944 of the Sos protein.

These artificial α-helices are expected to competitively interfere withSos helices for binding with Ras, thereby modulating the interaction ofRas and Sos.

By way of example, the artificial a-helices of the present invention canmimic at least a portion of the Sos protein as shown in Table 1.

TABLE 1 Exemplary Ras/Sos Helices. % Inhi- Name Sequence Solubilitybition wt (Sos929-944) FFGIYLTNILKTEEGN insoluble <10  1FEGIYRTDILRTEEGN partially 13 HBS 1 FEGIYRTDILRTEEGN partially 11  2FGEGIYRTDILRTEEGN partially <10  3 AEGIYRTDILRTEEGN partially <10  4AEGIYRADILRTEEGN partially <10  5 FEGIYRTDILR soluble <10  6FEGIYRTELLKAEEAN soluble 20 HBS 6 FEGIYRTELLKAEEAN soluble 40  7FEGIYRLELLKAEEAN soluble 37 HBS 7 FEGIYRLELLKAEEAN soluble 64HBS 7^(mut) AEGIYRLELLKAEAAA soluble 15  8 FEGIYRLELLK soluble <5 HBS 8FEGIYRLELLK soluble <5 HBS 9 FEGLLRLWLRKAibEEAN soluble 35 10FEGLLRLWLRKAibEEAibN soluble 50 HBS 10 FEGLLRLWLRKAibEEAibN soluble 6011 FEGIYRLELLKAibEEAibN soluble 30 HBS 11 FEGIYRLELLKAibEEAibN soluble20 12 FEGLLRLWLRKAEEAN soluble 50 HBS 12 FEGLLRLWLRKAEEAN soluble 55 13FEAIYRLELLKAEEAN soluble 40 HBS 13 FEAIYRLELLKAEEAN soluble 50 15FEAIYRLEKLKAEEAN soluble 50 HBS 15{circumflex over ( )}FEAIYRLEK*LKAE*EAN soluble <10 HBS 16 FEGIYRLEKLKAEEANRR soluble 56

Design of peptides and HBS helices that mimic Sos₉₂₉₋₉₄₄ α-H sequence.“^” refers to peptides containing a lactam-bridge between K* and E*residues (e.g. HBS 15). “%Inhibition” represents inhibition of theSos-mediated nucleotide exchange from Ras. Value is normalized for theexchange of nucleotide from Ras in the presence and absence of Sos.

Generally, suitable peptides of the present invention include those thatinclude the formula:

where

-   is a single or double carbon-carbon bond;-   is a single bond and is cis or trans when    is a double bond;-   n is 1 or 2;-   m is zero or any positive integer;-   R₁, R₂, R₃ and R₄ are independently hydrogen, an amino acid side    chain, an alkyl group, or an aryl group.

The variable m can be zero or any positive integer, for example 1, 2, 3,4 or 5. In some embodiments, m is 0, 1 or 2. In some embodiments, m is0. In other embodiments, m is 1 or 2.

The variable n can be 1 or 2. In other embodiments, n is 1. In stillother embodiments, n is 2.

The substituents R₁, R₂, R₃ and R₄ are independently hydrogen, an aminoacid side chain, an alkyl group, or an aryl group. For example, R₁, R₂,R₃ and R₄ are amino acid side chains. In some embodiments, R₁, R₂, R₃and R₄ are naturally occurring amino acid side chains. In otherembodiments, at least one amino acid side chain is a nonnaturallyoccurring side chain.

In some embodiments, R₃ is a peptide comprising a sequence of theformula FXGZZXZXZLXZEXXN (SEQ ID NO: 1) where X is any amino acidresidue and Z is a hydrophobic residue. In another embodiment, a peptideof the present invention includes an amino acid sequence of Table 1, andhas an internally-constrained α-helical region spanning residues 1through 4 of an amino acid sequence of Table 1.

As will be apparent to one of ordinary skill in the art, the methods ofthe present invention may be used to prepare peptides having highlystabilized, internally-constrained α-helices. The constraint may beplaced anywhere within the peptide, not just at the N-terminus. Forexample, a compound prepared according to the methods of the presentinvention may have the formula

The peptides produced according to the methods of the present inventionmay, for example, be less than 40, 30, 25, 20, or 15 amino acids,including, for example, less than 10 amino acid residues.

The present invention also relates to peptides having one or morestable, internally-constrained α-helices. The one or more stable,internally-constrained secondary structures includes the followingmotifs:

where

is a single or double bond,

is a single bond and is cis or trans when

is a double bond; n is 1 or 2; and m is any number. Examples of suchmotifs include:

HBS α-helices of the present invention are obtained by replacing anN-terminal main-chain i and i+4 hydrogen bond with a carbon-carbon bondthrough a ring-closing metathesis reaction, as shown in FIG. 2 (U.S.Pat. No. 7,202,332 to Arora et al.; Chapman & Arora, “OptimizedSynthesis of Hydrogen-bond Surrogate Helices: Surprising Effects ofMicrowave Heating on the Activity of Grubbs Catalysts,” Org. Lett.8:5825-8 (2006); Chapman et al., “A Highly Stable Short α-HelixConstrained by a Main-chain Hydrogen-bond Surrogate,” J. Am. Chem. Soc.126:12252-3 (2004); Dimartino et al., “Solid-phase Synthesis ofHydrogen-bond Surrogate-derived α-Helices,” Org. Lett. 7:2389-92 (2005),which are hereby incorporated by reference in their entirety). Thehydrogen bond surrogate pre-organizes an α-turn and stabilizes thepeptide sequence in an α-helical conformation. HBS α-helices have beenshown to adopt stable α-helical conformations from a variety of shortpeptide sequences (Wang et al., “Evaluation of Biologically RelevantShort α-Helices Stabilized by a Main-chain Hydrogen-bond Surrogate,” J.Am. Chem. Soc. 128:9248-56 (2006), which is hereby incorporated byreference in its entirety). It has also been shown that these artificialα-helices can target their expected protein receptor with high affinity(Wang et al., “Enhanced Metabolic Stability and Protein-bindingProperties of Artificial a Helices Derived from a Hydrogen-bondSurrogate: Application to Bcl-xL,” Angew. Chem. Int'l Ed. Engl.44:6525-9 (2005), originally published at Angew. Chem. 117:6683-7(2005), which is hereby incorporated by reference in its entirety).

In another aspect, preparing a compound of the invention involvesproviding a peptide precursor compound and promoting carbon-carbon bondformation to result in a stable, internally-constrained alpha-helix.

In one embodiment, the precursor has the formula:

The compound of the formula above may be reacted under conditionseffective to promote formation of a carbon-carbon bond. Such a reactionmay be, for example, metathesis. The exceptional functional grouptolerance displayed by the olefin metathesis catalysts for the facileintroduction of non-native carbon-carbon constraints in the preparationof peptidomimetics suggests that X and Y could be two carbon atomsconnected through an olefin metathesis reaction, as shown in Scheme 2(Hoveyda et al., “Ru Complexes Bearing Bidentate Carbenes: From InnocentCuriosity to Uniquely Effective Catalysts for Olefin Metathesis,” Org.Biomolec. Chem. 2:8-23 (2004); Trnka et al., “The Development ofL2X2Tu=CHR Olefin Metathesis Catalysts: An Organometallic SuccessStory,” Accounts Chem. Res. 34:18-29 (2001), which are herebyincorporated by reference in their entirety).

This aspect of the present invention may, for example, involve aring-closing olefin metathesis reaction. An olefin metathesis reactioncouples two double bonds (olefins) to afford two new double bonds (oneof which is typically ethylene gas). A ring-closing olefin metathesisutilizes an olefin metathesis reaction to form a macrocycle. In thisreaction, two double bonds within a chain are connected. The reactionmay be performed with a metathesis catalyst, for example of the formula

In other embodiments, the metathesis catalyst is of the formula

The metathesis reaction may be performed, for example, at a temperaturebetween about 25° C. and 110° C., and more preferably, at a temperatureof about 50° C.

The metathesis reaction may be performed with an organic solvent, suchas dichloromethane, dichloroethane, trichloroethane, or toluene.

The reactions disclosed herein may, for example, be carried out on asolid support. Suitable solid supports include particles, strands,precipitates, gels, sheets, tubing, spheres, containers, capillaries,pads, slices, films, plates, slides, discs, membranes, etc. These solidsupports can be made from a wide variety of materials, includingpolymers, plastics, ceramics, polysaccharides, silica or silica-basedmaterials, carbon, metals, inorganic glasses, membranes, or compositesthereof. The substrate is preferably flat but may take on a variety ofalternative surface configurations. For example, the substrate maycontain raised or depressed regions on which the synthesis takes place.The substrate and its surface preferably form a rigid support on whichto carry out the reactions described herein. Other substrate materialswill be readily apparent to those of ordinary skill in the art uponreview of this disclosure.

The metathesis reaction performed may initially yield a compound inwhich the newly formed carbon-carbon bond is a double bond. This doublebond can be subsequently converted to a single bond by hydrogenationmethods known in the art.

In another aspect, the invention provides a method of inhibiting Rassignaling in a cell, comprising contacting the cell with an effectiveamount of a composition comprising a peptide having a stable,internally-constrained alpha-helix, wherein said alpha helix isconstrained by a crosslink formed by a carbon-carbon bond-formingreaction, and further wherein the peptide mimics at least a portion of aprotein capable of interacting with a Ras protein. In some embodiments,the cell is in a living organism. The cell may be, for example, a cancercell such as a liquid or solid tumor cell. The invention furtherprovides a method of treating cancer in a subject in need thereof,comprising administering to the subject a peptide having a stable,internally-constrained alpha-helix, wherein said alpha helix isconstrained by a crosslink formed by a carbon-carbon bond-formingreaction, and further wherein the peptide mimics at least a portion of aprotein capable of interacting with a Ras protein.

As will be apparent to one of ordinary skill in the art, administeringmay be carried out using generally known methods.

Administration can be accomplished either via systemic administration tothe subject or via targeted administration to affected cells. Exemplaryroutes of administration include, without limitation, by intratrachealinoculation, aspiration, airway instillation, aerosolization,nebulization, intranasal instillation, oral or nasogastric instillation,intraperitoneal injection, intravascular injection, topically,transdermally, parenterally, subcutaneously, intravenous injection,intra-arterial injection (such as via the pulmonary artery),intramuscular injection, intrapleural instillation, intraventricularly,intralesionally, by application to mucous membranes (such as that of thenose, throat, bronchial tubes, genitals, and/or anus), or implantationof a sustained release vehicle.

Typically, the peptide of the present invention will be administered toa mammal as a pharmaceutical formulation that includes the therapeuticagent and any pharmaceutically acceptable adjuvants, carriers,excipients, and/or stabilizers, and can be in solid or liquid form, suchas tablets, capsules, powders, solutions, suspensions, or emulsions. Thecompositions preferably contain from about 0.01 to about 99 weightpercent, more preferably from about 2 to about 60 weight percent, oftherapeutic agent together with the adjuvants, carriers and/orexcipients. The amount of active compound in such therapeutically usefulcompositions is such that a suitable dosage unit will be obtained.

The agents may be orally administered, for example, with an inertdiluent, or with an assimilable edible carrier, or they may be enclosedin hard or soft shell capsules, or they may be compressed into tablets,or they may be incorporated directly with the food of the diet. For oraltherapeutic administration, these active compounds may be incorporatedwith excipients and used in the form of tablets, capsules, elixirs,suspensions, syrups, and the like. Such compositions and preparationsshould contain at least 0.1% of the agent. The percentage of the agentin these compositions may, of course, be varied and may conveniently bebetween about 2% to about 60% of the weight of the unit. The amount ofthe agent in such therapeutically useful compositions is such that asuitable dosage will be obtained.

The tablets, capsules, and the like may also contain a binder such asgum tragacanth, acacia, corn starch, or gelatin; excipients such asdicalcium phosphate; a disintegrating agent such as corn starch, potatostarch, or alginic acid; a lubricant such as magnesium stearate; and asweetening agent such as sucrose, lactose, or saccharin. When the dosageunit form is a capsule, it may contain, in addition to materials of theabove type, a liquid carrier, such as a fatty oil.

Various other materials may be present as coatings or to modify thephysical form of the dosage unit. For instance, tablets may be coatedwith shellac, sugar, or both. A syrup may contain, in addition to activeingredient(s), sucrose as a sweetening agent, methyl and propylparabensas preservatives, a dye, and flavoring such as cherry or orange flavor.

The agents may also be administered parenterally. Solutions orsuspensions of the agent can be prepared in water suitably mixed with asurfactant, such as hydroxypropylcellulose. Dispersions can also beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofin oils. Illustrative oils are those of petroleum, animal, vegetable, orsynthetic origin, for example, peanut oil, soybean oil, or mineral oil.In general, water, saline, aqueous dextrose and related sugar solutions,and glycols such as propylene glycol or polyethylene glycol, arepreferred liquid carriers, particularly for injectable solutions. Underordinary conditions of storage and use, these preparations contain apreservative to prevent the growth of microorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases, the form must be sterile and must be fluid tothe extent that easy syringability exists. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms, such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquidpolyethylene glycol), suitable mixtures thereof, and vegetable oils.

The agents according to this aspect of the present invention may also beadministered directly to the airways in the form of an aerosol. For useas aerosols, the compounds of the present invention in solution orsuspension may be packaged in a pressurized aerosol container togetherwith suitable propellants, for example, hydrocarbon propellants likepropane, butane, or isobutane with conventional adjuvants. The materialsof the present invention also may be administered in a non-pressurizedform such as in a nebulizer or atomizer.

The agents of the present invention may be administered directly to atargeted tissue, e.g., tissue that is susceptible to the condition to betreated. Additionally and/or alternatively, the agent may beadministered to a non-targeted area along with one or more agents thatfacilitate migration of the agent to (and/or uptake by) a targetedtissue, organ, or cell. As will be apparent to one of ordinary skill inthe art, the therapeutic agent itself be modified to facilitate itstransport to (and uptake by) the desired tissue, organ, or cell.

Exemplary delivery devices include, without limitation, nebulizers,atomizers, liposomes, transdermal patches, implants, implantable orinjectable protein depot compositions, and syringes. Other deliverysystems which are known to those of skill in the art can also beemployed to achieve the desired delivery of the therapeutic agent to thedesired organ, tissue, or cells in vivo to effect this aspect of thepresent invention.

Any suitable approach for delivery of the agents can be utilized topractice this aspect of the present invention. Typically, the agent willbe administered to a patient in a vehicle that delivers the agent(s) tothe target cell, tissue, or organ.

One approach for delivering agents into cells involves the use ofliposomes. Basically, this involves providing a liposome which includesagent(s) to be delivered, and then contacting the target cell, tissue,or organ with the liposomes under conditions effective for delivery ofthe agent into the cell, tissue, or organ.

Liposomes are vesicles comprised of one or more concentrically orderedlipid bilayers which encapsulate an aqueous phase. They are normally notleaky, but can become leaky if a hole or pore occurs in the membrane, ifthe membrane is dissolved or degrades, or if the membrane temperature isincreased to the phase transition temperature. Current methods of drugdelivery via liposomes require that the liposome carrier ultimatelybecome permeable and release the encapsulated drug at the target site.This can be accomplished, for example, in a passive manner where theliposome bilayer degrades over time through the action of various agentsin the body. Every liposome composition will have a characteristichalf-life in the circulation or at other sites in the body and, thus, bycontrolling the half-life of the liposome composition, the rate at whichthe bilayer degrades can be somewhat regulated.

In contrast to passive drug release, active drug release involves usingan agent to induce a permeability change in the liposome vesicle.Liposome membranes can be constructed so that they become destabilizedwhen the environment becomes acidic near the liposome membrane (see,e.g., Wang & Huang, “pH-Sensitive Immunoliposomes MediateTarget-cell-specific Delivery and Controlled Expression of a ForeignGene in Mouse,” Proc. Nat'l Acad. Sci. USA 84:7851-5 (1987), which ishereby incorporated by reference in its entirety). When liposomes areendocytosed by a target cell, for example, they can be routed to acidicendosomes which will destabilize the liposome and result in drugrelease.

Alternatively, the liposome membrane can be chemically modified suchthat an enzyme is placed as a coating on the membrane, which enzymeslowly destabilizes the liposome. Since control of drug release dependson the concentration of enzyme initially placed in the membrane, thereis no real effective way to modulate or alter drug release to achieve“on demand” drug delivery. The same problem exists for pH-sensitiveliposomes in that as soon as the liposome vesicle comes into contactwith a target cell, it will be engulfed and a drop in pH will lead todrug release.

This liposome delivery system can also be made to accumulate at a targetorgan, tissue, or cell via active targeting (e.g., by incorporating anantibody or hormone on the surface of the liposomal vehicle). This canbe achieved according to known methods.

Different types of liposomes can be prepared according to Bangham etal., “Diffusion of Univalent Ions Across the Lamellae of SwollenPhospholipids,”J. Mol. Biol. 13:238-52 (1965); U.S. Pat. No. 5,653,996to Hsu; U.S. Pat. No. 5,643,599 to Lee et al.; U.S. Pat. No. 5,885,613to Holland et al.; U.S. Pat. No. 5,631,237 to Dzau & Kaneda; and U.S.Pat. No. 5,059,421 to Loughrey et al., each of which is herebyincorporated by reference in its entirety.

These liposomes can be produced such that they contain, in addition tothe therapeutic agents of the present invention, other therapeuticagents, such as anti-inflammatory agents, which would then be releasedat the target site (e.g., Wolff et al., “The Use of Monoclonal Anti-Thy1IgG1 for the Targeting of Liposomes to AKR-A Cells in Vitro and inVivo,” Biochim. Biophys. Acta 802:259-73 (1984), which is herebyincorporated by reference in its entirety).

An alternative approach for delivery of proteins or polypeptide agents(e.g., peptides of the present invention) involves the conjugation ofthe desired protein or polypeptide to a polymer that is stabilized toavoid enzymatic degradation of the conjugated protein or polypeptide.Conjugated proteins or polypeptides of this type are described in U.S.Pat. No. 5,681,811 to Ekwuribe, which is hereby incorporated byreference in its entirety.

Yet another approach for delivery of proteins or polypeptide agentsinvolves preparation of chimeric proteins according to U.S. Pat. No.5,817,789 to Heartlein et al., which is hereby incorporated by referencein its entirety. The chimeric protein can include a ligand domain andthe polypeptide agent (e.g., the artificial α-helix of the presentinvention). The ligand domain is specific for receptors located on atarget cell. Thus, when the chimeric protein is delivered intravenouslyor otherwise introduced into blood or lymph, the chimeric protein willadsorb to the targeted cell, and the targeted cell will internalize thechimeric protein.

Administration can be carried out as frequently as required and for aduration that is suitable to provide effective treatment. For example,administration can be carried out with a single sustained-release dosageformulation or with multiple daily doses.

The amount to be administered will, of course, vary depending upon thetreatment regimen. Generally, an agent is administered to achieve anamount effective for an improvement in the state of the patient (i.e., atherapeutically effective amount). Thus, in the case of cancer, atherapeutically effective amount can be an amount which is capable of atleast partially decreasing the size of a tumor, decreasing the number ofcancerous cells in the body, or slowing the increase in number of cancercells in the body. The dose required to obtain an effective amount mayvary depending on the agent, formulation, cancer, and individual to whomthe agent is administered.

Determination of effective amounts may also involve in vitro assays inwhich varying doses of agent are administered to cells in culture andthe concentration of agent effective for inhibiting growth of cancercells is determined in order to calculate the concentration required invivo. Effective amounts may also be based on in vivo animal studies. Atherapeutically effective amount can be determined empirically by thoseof skill in the art.

Methods of Treatment

In some embodiments, the compounds of the invention is used to treat,prevent, and/or diagnose cancers and neoplastic conditions. As usedherein, the terms “cancer”, “hyperproliferative” and “neoplastic” referto cells having the capacity for autonomous growth, i.e., an abnormalstate or condition characterized by rapidly proliferating cell growth.Hyperproliferative and neoplastic disease states may be categorized aspathologic, i.e., characterizing or constituting a disease state, or maybe categorized as non-pathologic, i.e., a deviation from normal but notassociated with a disease state. The term is meant to include all typesof cancerous growths or oncogenic processes, metastatic tissues ormalignantly transformed cells, tissues, or organs, irrespective ofhistopathologic type or stage of invasiveness. A metastatic tumor canarise from a multitude of primary tumor types, including but not limitedto those of breast, lung, liver, colon and ovarian origin. “Pathologichyperproliferative” cells occur in disease states characterized bymalignant tumor growth. Examples of non-pathologic hyperproliferativecells include proliferation of cells associated with wound repair.Examples of cellular proliferative and/or differentiative disordersinclude cancer, e.g., carcinoma, sarcoma, or metastatic disorders. Insome embodiments, the compounds are novel therapeutic agents forcontrolling breast cancer, ovarian cancer, colon cancer, lung cancer,metastasis of such cancers and the like.

Examples of cancers or neoplastic conditions include, but are notlimited to, a fibrosarcoma, myosarcoma, liposarcoma, chondrosarcoma,osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma,lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma,Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, gastric cancer,esophageal cancer, rectal cancer, pancreatic cancer, ovarian cancer,prostate cancer, uterine cancer, cancer of the head and neck, skincancer, brain cancer, squamous cell carcinoma, sebaceous glandcarcinoma, papillary carcinoma, papillary adenocarcinoma,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, testicularcancer, small cell lung carcinoma, non-small cell lung carcinoma,bladder carcinoma, epithelial carcinoma, glioma, astrocytoma,medulloblastoma, craniopharyngioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma,melanoma, neuroblastoma, retinoblastoma, leukemia, lymphoma, or Kaposisarcoma.

Examples of proliferative disorders include hematopoietic neoplasticdisorders. As used herein, the term “hematopoietic neoplastic disorders”includes diseases involving hyperplastic/neoplastic cells ofhematopoietic origin, e.g., arising from myeloid, lymphoid or erythroidlineages, or precursor cells thereof. Preferably, the diseases arisefrom poorly differentiated acute leukemias, e.g., erythroblasticleukemia and acute megakaryoblastic leukemia. Additional exemplarymyeloid disorders include, but are not limited to, acute promyeloidleukemia (APML), acute myelogenous leukemia (AML) and chronicmyelogenous leukemia (CML) (reviewed in Vaickus (1991), Cril Rev.Oncol./Hemotol. 11:267-97); lymphoid malignancies include, but are notlimited to acute lymphoblastic leukemia (ALL) which includes B-lineageALL and T-lineage ALL, chronic lymphocytic leukemia (CLL),prolymphocytic leukemia (PLL), hairy cell leukemia (HLL) andWaldenstrom's macroglobulinemia (WM). Additional forms of malignantlymphomas include, but are not limited to non-Hodgkin lymphoma andvariants thereof, peripheral T cell lymphomas, adult T cellleukemia/lymphoma (ATL), cutaneous T-cell lymphoma (CTCL), largegranular lymphocytic leukemia (LGF), Hodgkin's disease and Reed-Stembergdisease.

Examples of cellular proliferative and/or differentiative disorders ofthe breast include, but are not limited to, proliferative breast diseaseincluding, e.g., epithelial hyperplasia, sclerosing adenosis, and smallduct papillomas; tumors, e.g., stromal tumors such as fibroadenoma,phyllodes tumor, and sarcomas, and epithelial tumors such as large ductpapilloma; carcinoma of the breast including in situ (noninvasive)carcinoma that includes ductal carcinoma in situ (including Paget'sdisease) and lobular carcinoma in situ, and invasive (infiltrating)carcinoma including, but not limited to, invasive ductal carcinoma,invasive lobular carcinoma, medullary carcinoma, colloid (mucinous)carcinoma, tubular carcinoma, and invasive papillary carcinoma, andmiscellaneous malignant neoplasms. Disorders in the male breast include,but are not limited to, gynecomastia and carcinoma.

Examples of cellular proliferative and/or differentiative disorders ofthe lung include, but are not limited to, bronchogenic carcinoma,including paraneoplastic syndromes, bronchioloalveolar carcinoma,neuroendocrine tumors, such as bronchial carcinoid, miscellaneoustumors, and metastatic tumors; pathologies of the pleura, includinginflammatory pleural effusions, noninflammatory pleural effusions,pneumothorax, and pleural tumors, including solitary fibrous tumors(pleural fibroma) and malignant mesothelioma.

Examples of cellular proliferative and/or differentiative disorders ofthe colon include, but are not limited to, non-neoplastic polyps,adenomas, familial syndromes, colorectal carcinogenesis, colorectalcarcinoma, and carcinoid tumors.

Examples of cellular proliferative and/or differentiative disorders ofthe liver include, but are not limited to, nodular hyperplasias,adenomas, and malignant tumors, including primary carcinoma of the liverand metastatic tumors.

Examples of cellular proliferative and/or differentiative disorders ofthe ovary include, but are not limited to, ovarian tumors such as,tumors of coelomic epithelium, serous tumors, mucinous tumors,endometrioid tumors, clear cell adenocarcinoma, cystadenofibroma,Brenner tumor, surface epithelial tumors; germ cell tumors such asmature (benign) teratomas, monodermal teratomas, immature malignantteratomas, dysgerminoma, endodermal sinus tumor, choriocarcinoma; sexcord-stomal tumors such as, granulosa-theca cell tumors,thecomafibromas, androblastomas, hill cell tumors, and gonadoblastoma;and metastatic tumors such as Krukenberg tumors.

In some embodiments, the peptides of the invention are used to treat acancer mediated by a mutated Ras protein. Cancers known to frequentlyinvolve such mutations include, but are not limited to, non-small-celllung cancer (adenocarcinoma), colorectal cancer, pancreatic cancer,thyroid cancers (e.g. follicular, undifferentiated papillary orpapillary), seminoma, melanoma, bladder cancer, liver cancer, kidneycancer, myelodysplastic syndrome, and acute myelogenous leukemia.

Breast Cancer

In one aspect, the invention provides methods of treating breast cancerby administering the compounds of the invention. Breast cancer includesinvasive breast carcinomas, such as invasive ductal carcinoma, invasivelobular carcinoma, tubular carcinoma, invasive cribriform carcinoma,medullary carcinoma, mucinous carcinoma and other tumours with abundantmucin, cystadenocarcinoma, columnar cell mucinous carcinoma, signet ringcell carcinoma, neuroendocrine tumours (including solid neuroendocrinecarcinoma, atypical carcinoid tumour, small cell/oat cell carcinoma, orlarge cell neuroendocrine carcioma), invasive papillary carcinoma,invasive micropapillary carcinoma, apocrine carcinoma, metaplasticcarcinomas, pure epithelial metaplastic carciomas, mixedepithelial/mesenchymal metaplastic carcinomas, lipid-rich carcinoma,secretory carcinoma, oncocytic carcinoma, adenoid cystic carcinoma,acinic cell carcinoma, glycogen-rich clear cell carcinoma, sebaceouscarcinoma, inflammatory carcinoma or bilateral breast carcinoma;mesenchymal tumors such as haemangioma, angiomatosis,haemangiopericytoma, pseudoangiomatous stromal hyperplasia,myofibroblastoma, fibromatosis (aggressive), inflammatorymyofibroblastic tumour, lipoma, angiolipoma, granular cell tumour,neurofibroma, schwannoma, angiosarcoma, liposarcoma, rhabdomyosarcoma,osteosarcoma, leiomyoma, or leiomysarcoma; myoepithelial lesions such asmyoepitheliosis, adenomyoepithelial adenosis, adenomyoepithelioma, ormalignant myoepithelioma; fibroepithelial tumours such as fibroadenoma,phyllodes tumour, low grade periductal stromal sarcoma, or mammaryhamartoma; and tumours of the nipple such as nipple adenoma,syringomatous adenoma, or Paget's disease of the nipple.

Treatment of breast cancer may be effected in conjunction with anyadditional therapy, such as a therapy that is part of the standard ofcare. A surgical technique such as lumpectomy or mastectomy may beperformed prior to, during, or following treatment with the compounds ofthe invention. Alternatively, radiation therapy may be used for thetreatment of breast cancer in conjunction with the compounds of theinvention. In other cases, the compounds of the invention areadministered in combination with a second therapeutic agent. Such anagent may be a chemotherapeutic agent such as an individual drug orcombination of drugs and therapies. For example, the chemotherapeuticagent can be an adjuvant chemotherapeutic treatment such as CMF(cyclophosphamide, methotrexate, and 5-fluorouracil); FAC or CAF(5-fluorouracil, doxorubicin, cyclophosphamide); AC or CA (doxorubicinand cyclophosphamide); AC-Taxol (AC followed by paclitaxel); TAC(docetaxel, doxorubicin, and cyclophosphamide); FEC (5-fluorouracil,epirubicin and cyclophosphamide); FECD (FEC followed by docetaxel); TC(docetaxel and cyclophosphamide). In addition to chemotherapy,trastuzumab may also be added to the regimen depending on the tumorcharacteristics (i.e. HER2/neu status) and risk of relapse. Hormonaltherapy may also be appropriate before, during or followingchemotherapeutic treatment. For example, tamoxifen may be administeredor a compound in the category of aromatase inhibitors including, but notlimited to aminogluthetimide, anastrozole, exemestane, formestane,letrozole, or vorozole. In other embodiments, an antiangiogenic agentmay be used in combination therapy for the treatment of breast cancer.The antiangiogenic agent may be an anti-VEGF agent including, but notlimited to bevacizumab.

Ovarian Cancer

In another aspect, the compounds of the invention may be used to treatovarian cancer. Ovarian cancers include ovarian tumors such as, tumorsof coelomic epithelium, serous tumors, mucinous tumors, endometrioidtumors, clear cell adenocarcinoma, cystadenofibroma, Brenner tumor,surface epithelial tumors; germ cell tumors such as mature (benign)teratomas, monodermal teratomas, immature malignant teratomas,dysgerminoma, endodermal sinus tumor, choriocarcinoma; sex cord-stomaltumors such as, granulosa-theca cell tumors, thecomafibromas,androblastomas, hill cell tumors, and gonadoblastoma; and metastatictumors such as Krukenberg tumors.

The compounds of the invention may be administered in conjunction with asecond therapy such as a therapy that is part of the standard of care.Surgery, immunotherapy, chemotherapy, hormone therapy, radiationtherapy, or a combination thereof are some possible treatments availablefor ovarian cancer. Some possible surgical procedures include debulking,and a unilateral or bilateral oophorectomy and/or a unilateral orbilateral salpigectomy.

Anti-cancer drugs that may be used include cyclophosphamide, etoposide,altretamine, and ifosfamide. Hormone therapy with the drug tamoxifen maybe used to shrink ovarian tumors. Radiation therapy may be external beamradiation therapy and/or brachytherapy.

Prostate Cancer

In another aspect, the compounds of the invention may be used to treatprostate cancer. Prostate cancers include adenocarcinomas andmetastasized adenocarcinomas. The compounds of the invention may beadministered in conjunction with a second therapy such as a therapy thatis part of the standard of care. Treatment for prostate cancer mayinvolve surgery, radiation therapy, High Intensity Focused Ultrasound(HIFU), chemotherapy, cryosurgery, hormonal therapy, or any combinationthereof. Surgery may involve prostatectomy, radical perinealprostatectomy, laparoscopic radical prostatectomy, transurethralresection of the prostate or orchiectomy. Radiation therapy may includeexternal beam radiation therapy and/or brachytherapy. Hormonal therapymay include orchiectomy; administration of antiandrogens such asflutamide, bicalutamide, nilutamide, or cyproterone acetate; medicationswhich inhibit the production of adrenal androgens such as DHEA, such asketoconazole and aminoglutethimide; and GnRH antagonists or agonistssuch as Abarelix (Plenaxis®), Cetrorelix (Cetrotide®), Ganirelix(Antagon®), leuprolide, goserelin, triptorelin, or buserelin. Treatmentwith an anti-androgen agent, which blocks androgen activity in the body,is another available therapy. Such agents include flutamide,bicalutamide, and nilutamide. This therapy is typically combined withLHRH analog administration or an orchiectomy, which is termed a combinedandrogen blockade (CAB). Chemotherapy includes, but is not limited to,administration of docetaxel, for example with a corticosteroid such asprednisone. Anti-cancer drugs such as doxorubicin, estramustine,etoposide, mitoxantrone, vinblastine, paclitaxel, carboplatin may alsobe administered to slow the growth of prostate cancer, reduce symptomsand improve the quality of life. Additional compounds such asbisphosphonate drugs may also be administered.

Renal Cancer

In another aspect, the compounds of the invention may be used to treatrenal cancer. Renal cancers include, but are not limited to, renal cellcarcinomas, metastases from extra-renal primary neoplasms, renallymphomas, squamous cell carcinomas, juxtaglomerular tumors (reninomas),transitional cell carcinomas, angiomyolipomas, oncocytomas and Wilm'stumors. The compounds of the invention may be administered inconjunction with a second therapy such as a therapy that is part of thestandard of care. Treatment for renal cancer may involve surgery,percutaneous therapies, radiation therapies, chemotherapy, vaccines, orother medication. Surgical techniques useful for treatment of renalcancer in combination with the compounds of the invention includenephrectomy, which may include removal of the adrenal gland,retroperitoneal lymph nodes, and any other surrounding tissues affectedby the invasion of the tumor. Percutaneous therapies include, forexample, image-guided therapies which may involve imaging of a tumorfollowed by its targeted destruction by radiofrequency ablation orcryotherapy. In some cases, other chemotherapeutic or other medicationsuseful in treating renal cancer may be alpha-interferon, interleukin-2,bevacizumab, sorafenib, sunitib, temsirolimus or other kinaseinhibitors.

Pancreatic Cancer

In other aspects, the invention provides methods of treating pancreaticcancer by administering compounds of the invention, such as a pancreaticcancer selected from the following: an epitheliod carcinoma in thepancreatic duct tissue and an adenocarcinoma in a pancreatic duct. Themost common type of pancreatic cancer is an adenocarcinoma, which occursin the lining of the pancreatic duct. Possible treatments available forpancreatic cancer include surgery, immunotherapy, radiation therapy, andchemotherapy. Possible surgical treatment options include a distal ortotal pancreatectomy and a pancreaticoduodenectomy (Whipple procedure).Radiation therapy may be an option for pancreatic cancer patients,specifically external beam radiation where radiation is focused on thetumor by a machine outside the body. Another option is intraoperativeelectron beam radiation administered during an operation. Chemotherapymay also be used to treat pancreatic cancer patients. Suitableanti-cancer drugs include, but are not limited to, 5-fluorouracil(5-FU), mitomycin, ifosfamide, doxorubicin, streptozocin, chlorozotocin,and combinations thereof. The methods provided by the invention canprovide a beneficial effect for pancreatic cancer patients, byadministration of a polypeptide of the invention or a combination ofadministration of a compound and surgery, radiation therapy, orchemotherapy.

Colon Cancer

In one aspect, compounds of the invention may be used for the treatmentof colon cancer, including but not limited to non-neoplastic polyps,adenomas, familial syndromes, colorectal carcinogenesis, colorectalcarcinoma, and carcinoid tumors. Possible treatments available for coloncancer that may be used in conjunction with the compounds of theinvention include surgery, chemotherapy, radiation therapy or targeteddrug therapy.

Radiation therapy may include external beam radiation therapy and/orbrachytherapy. Chemotherapy may be used to reduce the likelihood ofmetastasis developing, shrink tumor size, or slow tumor growth.Chemotherapy is often applied after surgery (adjuvant), before surgery(neo-adjuvant), or as the primary therapy if surgery is not indicated(palliative). For example, exemplary regimens for adjuvant chemotherapyinvolve the combination of infusional 5-fluorouracil, leucovorin, andoxaliplatin (FOLFOX). First line chemotherapy regimens may involve thecombination of infusional 5-fluorouracil, leucovorin, and oxaliplatin(FOLFOX) with a targeted drug such as bevacizumab, cetuximab orpanitumumab or infusional 5-fluorouracil, leucovorin, and irinotecan(FOLFIRI) with targeted drug such as bevacizumab, cetuximab orpanitumumab. Other chemotherapeutic agents that may be useful in thetreatment or prevention of colon cancer in combination with thecompounds of the invention are Bortezomib (Velcade®), Oblimersen(Genasense®, G3139), Gefitinib and Erlotinib (Tarceva®) and Topotecan(Hycamtin®).

Lung Cancer

Some embodiments provide methods for the treatment of lung cancer usingthe compounds of the invention. Examples of cellular proliferativeand/or differentiative disorders of the lung include, but are notlimited to, bronchogenic carcinoma, including paraneoplastic syndromes,bronchioloalveolar carcinoma, neuroendocrine tumors, such as bronchialcarcinoid, miscellaneous tumors, and metastatic tumors; pathologies ofthe pleura, including inflammatory pleural effusions, noninflammatorypleural effusions, pneumothorax, and pleural tumors, including solitaryfibrous tumors (pleural fibroma) and malignant mesothelioma.

The most common type of lung cancer is non-small cell lung cancer(NSCLC), which accounts for approximately 80-85% of lung cancers and isdivided into squamous cell carcinomas, adenocarcinomas, and large cellundifferentiated carcinomas. Small cell lung cancer, e.g. small celllung carcinomas, accounts for 15-20% of lung cancers. Treatment optionsfor lung cancer include surgery, immunotherapy, radiation therapy,chemotherapy, photodynamic therapy, or a combination thereof. Somepossible surgical options for treatment of lung cancer are a segmentalor wedge resection, a lobectomy, or a pneumonectomy. Radiation therapymay be external beam radiation therapy or brachytherapy. Someanti-cancer drugs that may be used in chemotherapy to treat lung cancerin combination with the compounds of the invention include cisplatin,carboplatin, paclitaxel, docetaxel, gemcitabine, vinorelbine,irinotecan, etoposide, vinblastine, gefitinib, ifosfamide, methotrexate,or a combination thereof. Photodynamic therapy (PDT) may be used totreat lung cancer patients. The methods described herein can provide abeneficial effect for lung cancer patients, by administration of acompound or a combination of administration of a compound and surgery,radiation therapy, chemotherapy, photodynamic therapy, or a combinationthereof.

Examples of cellular proliferative and/or differentiative disorders ofthe liver include, but are not limited to, nodular hyperplasias,adenomas, and malignant tumors, including primary carcinoma of the liverand metastatic tumors.

Immunoproliferative Disorders

Immunoproliferative disorders (also known as “immunoproliferativediseases” or “immunoproliferative neoplasms”) are disorders of theimmune system that are characterized by the abnormal proliferation ofthe primary cells of the immune system, which includes B cells, T cellsand Natural Killer (NK) cells, or by the excessive production ofimmunoglobulins (also known as antibodies). Such disorders include thegeneral categories of lymphoproliferative disorders,hypergammaglobulinemias, and paraproteinemias. Examples of suchdisorders include, but are not limited to, X-linked lymphoproliferativedisorder, autosomal lymphoproliferative disorder, Hyper-IgM syndrome,heavy chain disease, and cryoglobulinemia. Other immunoproliferativedisorders can be graft versus host disease (GVHD); psoriasis; immunedisorders associated with graft transplantation rejection; T celllymphoma; T cell acute lymphoblastic leukemia; testicular angiocentric Tcell lymphoma; benign lymphocytic angiitis; and autoimmune diseases suchas lupus erythematosus, Hashimoto's thyroiditis, primary myxedema,Graves' disease, pernicious anemia, autoimmune atrophic gastritis,Addison's disease, insulin dependent diabetes mellitis, good pasture'ssyndrome, myasthenia gravis, pemphigus, Crohn's disease, sympatheticophthalmia, autoimmune uveitis, multiple sclerosis, autoimmune hemolyticanemia, idiopathic thrombocytopenia, primary biliary cirrhosis, chronicaction hepatitis, ulceratis colitis, Sjogren's syndrome, rheumatoidarthritis, polymyositis, scleroderma, and mixed connective tissuedisease.

Combination Treatments

In one embodiment, compounds of the invention may be used for thetreatment of cancer in conjunction with alkylating and alkylating-likeagents. Such agents include, for example, nitrogen mustards such aschlorambucil, chlormethine, cyclophosphamide, ifosfamide, and melphalan;nitrosoureas such as carmustine, fotemustine, lomustine, andstreptozocin; platinum therapeutic agents such as carboplatin,cisplatin, oxaliplatin, BBR3464, and satraplatin; or other agents,including but not limited to busulfan, dacarbazine, procarbazine,temozolomide, thiotepa, treosulfan, or uramustine.

In another embodiment, compounds of the invention may be used inconjunction with an antineoplastic agent which is an antimetabolite. Forexample, such an antineoplastic agent may be a folic acid such asaminopterin, methotrexate, pemetrexed, or raltitrexed. Alternatively,the antineoplastic agent may be a purine, including but not limited tocladribine, clofarabine, fludarabine, mercaptopurine, pentostatin,thioguanine. In further embodiments, the antineoplastic agent may be apyrimidine such as capecitabine, cytarabine, fluorouracil, Floxuridine,and gemcitabine.

In still other embodiments, compounds of the invention may be used inconjunction with an antineoplastic agent which is an spindlepoison/mitotic inhibitor. Agents in this category include taxanes, forexample docetaxel and paclitaxel; and vinca alkaloids such asvinblastine, vincristine, vindesine, and vinorelbine. In yet otherembodiments, compounds of the invention may be used in combination withan antineoplastic agent which is a cytotoxic/antitumor antibiotic fromthe anthracycline family such as daunorubicin, doxorubicin, epirubicin,idarubicin, mitoxantrone, pixantrone, or valrubicin; an antibiotic fromthe streptomyces family such as actinomycin, bleomycin, mitomycin, orplicamycin; or hydroxyurea. Alternatively, agents used for combinationtherapy may be topoisomerase inhibitors including, but not limited tocamptothecin, topotecan, irinotecan, etoposide, or teniposide.

Alternatively, the antineoplastic agent may be an antibody orantibody-derived agent. For example, a receptor tyrosine kinase-targetedantibody such as cetuximab, panitumumab, or trastuzumab may be usedAlternatively, the antibody may be an anti-CD20 antibody such asrituximab or tositumomab, or any other suitable antibody including butnot limited to alemtuzumab, bevacizumab, and gemtuzumab. In otherembodiments, the antineoplastic agent is a photosensitizer such asaminolevulinic acid, methyl aminolevulinate, porfimer sodium, orverteporfin. In still other embodiments, the antineoplastic agent is atyrosine kinase inhibitor such as dediranib, dasatinib, erlotinib,gefitinib, imatinib, lapatinib, nilotinib, sorafenib, sunitinib, orvandetanib. Other neoplastic agents suitable in the use of the inventioninclude, for example, alitretinoin, tretinoin, altretamine, amsacrine,anagrelide, arsenic trioxide, asparaginase (pegaspargase), bexarotene,bortezomib, denileukin diftitox, estramustine, ixabepilone, masoprocol,or mitotane.

In other or further embodiments, the compounds described herein are usedto treat, prevent or diagnose conditions characterized by overactivecell death or cellular death due to physiologic insult, etc. Someexamples of conditions characterized by premature or unwanted cell deathare or alternatively unwanted or excessive cellular proliferationinclude, but are not limited to hypocellular/hypoplastic,acellular/aplastic, or hypercellular/hyperplastic conditions. Someexamples include hematologic disorders including but not limited tofanconi anemia, aplastic anemia, thalaessemia, congenital neutropenia,and myelodysplasia.

In other or further embodiments, the compounds of the invention that actto decrease apoptosis are used to treat disorders associated with anundesirable level of cell death. Thus, in some embodiments, theanti-apoptotic compounds of the invention are used to treat disorderssuch as those that lead to cell death associated with viral infection,e.g., infection associated with infection with human immunodeficiencyvirus (HIV). A wide variety of neurological diseases are characterizedby the gradual loss of specific sets of neurons, and the anti-apoptoticcompounds of the invention are used, in some embodiments, in thetreatment of these disorders. Such disorders include Alzheimer'sdisease, Parkinson's disease, amyotrophic lateral sclerosis (ALS)retinitis pigmentosa, spinal muscular atrophy, and various forms ofcerebellar degeneration. The cell loss in these diseases does not inducean inflammatory response, and apoptosis appears to be the mechanism ofcell death. In addition, a number of hematologic diseases are associatedwith a decreased production of blood cells. These disorders includeanemia associated with chronic disease, aplastic anemia, chronicneutropenia, and the myelodysplastic syndromes. Disorders of blood cellproduction, such as myelodysplastic syndrome and some forms of aplasticanemia, are associated with increased apoptotic cell death within thebone marrow. These disorders could result from the activation of genesthat promote apoptosis, acquired deficiencies in stromal cells orhematopoietic survival factors, or the direct effects of toxins andmediators of immune responses. Two common disorders associated with celldeath are myocardial infarctions and stroke. In both disorders, cellswithin the central area of ischemia, which is produced in the event ofacute loss of blood flow, appear to die rapidly as a result of necrosis.However, outside the central ischemic zone, cells die over a moreprotracted time period and morphologically appear to die by apoptosis.

Other Methods of Use

In other or further embodiments, the anti-apoptotic compounds of theinvention are used to treat all such disorders associated withundesirable cell death.

Some examples of immunologic disorders that are treated with thecompounds described herein include but are not limited to organtransplant rejection, arthritis, lupus, IBD, Crohn's disease, asthma,multiple sclerosis, diabetes, etc.

Some examples of neurologic disorders that are treated with thecompounds described herein include but are not limited to Alzheimer'sDisease, Down's Syndrome, Dutch Type Hereditary Cerebral HemorrhageAmyloidosis, Reactive Amyloidosis, Familial Amyloid Nephropathy withUrticaria and Deafness, Muckle-Wells Syndrome, Idiopathic Myeloma;Macroglobulinemia-Associated Myeloma, Familial Amyloid Polyneuropathy,Familial Amyloid Cardiomyopathy, Isolated Cardiac Amyloid, SystemicSenile Amyloidosis, Adult Onset Diabetes, Insulinoma, Isolated AtrialAmyloid, Medullary Carcinoma of the Thyroid, Familial Amyloidosis,Hereditary Cerebral Hemorrhage With Amyloidosis, Familial AmyloidoticPolyneuropathy, Scrapie, Creutzfeldt-Jacob Disease, GerstmannStraussler-Scheinker Syndrome, Bovine Spongiform Encephalitis, aprion-mediated disease, and Huntington's Disease.

Some examples of endocrinologic disorders that are treated with thecompounds described herein include but are not limited to diabetes,hypothyroidism, hypopituitarism, hypoparathyroidism, hypogonadism, etc.

Examples of cardiovascular disorders (e.g., inflammatory disorders) thatare treated or prevented with the compounds of the invention include,but are not limited to, atherosclerosis, myocardial infarction, stroke,thrombosis, aneurism, heart failure, ischemic heart disease, anginapectoris, sudden cardiac death, hypertensive heart disease; non-coronaryvessel disease, such as arteriolosclerosis, small vessel disease,nephropathy, hypertriglyceridemia, hypercholesterolemia, hyperlipidemia,xanthomatosis, asthma, hypertension, emphysema and chronic pulmonarydisease; or a cardiovascular condition associated with interventionalprocedures (“procedural vascular trauma”), such as restenosis followingangioplasty, placement of a shunt, stent, synthetic or natural excisiongrafts, indwelling catheter, valve or other implantable devices.Preferred cardiovascular disorders include atherosclerosis, myocardialinfarction, aneurism, and stroke.

EXAMPLES Example 1 Synthesis of Peptides 1-17

Peptides 1-17 were synthesized as shown in FIGS. 12-15. Resin bound freeamine peptides were synthesized by conventional Fmoc solid-phasechemistry on Rink Amide or Knorr resin (loading=0. 4mmole/g) on a CEMLiberty Microwave Peptide Synthesizer. Standard Fmoc amino acids (and4-petenoic acid) (5 equiv) were activated with HBTU (4.9 equiv) in 6%DIPEA/NMP solution for 15 min and added to the resin bound free amine.The resulting mixture was shaken for 60 minutes. The coupling efficiencywas monitored by the ninhydrin test. Fmoc groups were deprotected bytreatment with 20% piperidine in NMP (2×20 min). The bis-olefin peptidecontaining resin was thoroughly washed with DMF and DCM respectively,and dried under vacuum overnight.

Microwave-assisted ring-closing metathesis reactions on resin-boundbis-olefins were performed with the Hoveyda-Grubbs catalyst (0.15equiv.) in dichloroethane as described in Chapman & Arora, “OptimizedSynthesis of Hydrogen-bond Surrogate Helices: Surprising Effects ofMicrowave Heating on the Activity of Grubbs Catalysts,” Org. Lett.8:5825-8 (2006), which is hereby incorporated by reference in itsentirety. The reaction mixture was irradiated with these settings: 250 Wmaximum power, 120° C., 5 minute ramp time, and 10 minute hold time.Resin bound peptides were cleaved from the resin by treatment with acleavage cocktail (CF₃CO₂H:H₂O:triisopropylsilane, 95:2.5:2.5) for 1.5hours, and purified by reversed-phase HPLC.

Several peptides of the invention were examined using liquidchromatography-mass spectrometry (“LCMS”). LCMS data were obtained on anAgilent 1100 series. The LCMS results are shown in Table 2.

TABLE 2 Mass spectrometry results for Peptides 1-12 (LC/MSD (XCT)electrospray trap). Mass Calculated Mass Observed Name Sequence [M]⁺[M]⁺ wt (Sos929-944) FFGIYLTNILKTEEGN 1900.1 1900.2  1 FEGIYRTDILRTEEGN1954.1 1955.1 HBS 1 FEGIYRTDILRTEEGN 2006.1 1003.7  2 FGEGIYRTDILRTEEGN2011.1 2012.1  3 AEGIYRTDILRTEEGN 1878.0 1878.1  4 AEGIYRADILRTEEGN1847.9 924.9*  5 FEGIYRTDILR 1423.6 1423.3  6 FEGIYRTELLKAEEAN 1924.11924.1 HBS 6 FEGIYRTELLKAEEAN 1976.1 1978.2  7 FEGIYRLELLKAEEAN 1936.1968.7 HBS 7 FEGIYRLELLKAEEAN 1988.2 1989.9 HBS 7^(mut) AEGIYRLELLKAEAAA1811.0 1813.3  8 FEGIYRLELLK 1422.6 711.6* HBS 8 FEGIYRLELLK 1473.71474.9  9 FEGLLRLWLRKAibEEAN 2014.3 672.2** HBS 9 FEGLLRLWLRKAibEEAN2052.3 684.9** 10 FEGLLRLWLRKAibEEAibN 2014.3 672.4** HBS 10FEGLLRLWLRKAibEEAibN 2066.4 689.7** 11 FEGIYRLELLKAibEEAibN 1964.21965.1 HBS 11 FEGIYRLELLKAibEEAibN 2016.2 1009.1* 12 FEGLLRLWLRKAEEAN1986.2 993.4* HBS 12 FEGLLRLWLRKAEEAN 2038.3 1019.5* 13 FEAIYRLELLKAEEAN1950.1 975.6* HBS 13 FEAIYRLELLKAEEAN 2001.2 2003.0 15 FEAIYRLEKLKAEEAN1965.2 1966.1 HBS 15 FEAIYRLEK^(#)LKAE^(#)EAN 1999.2 2000.1 HBS 16FEGIYRLEKLKAEEANRR 2343.6 2346.0 In Table 2, ^(#)represents a lactambridge between the lysine and glutamic acid residues. *represents [M]²⁺and ** represents [M]³⁺.

Fluoresceinated versions of exemplary HBS peptides of the invention werealso prepared and are shown below:

Example 2 Circular Dichroism Spectroscopy

CD spectra shown in FIG. 9 were recorded on an AVIV 202SF CDspectrometer equipped with a temperature controller using 1 mm lengthcells and a scan speed of 5 nm/min. The spectra were averaged over 10scans with the baseline subtracted from analogous conditions as that forthe samples. Samples were prepared in 0.1× phosphate buffered saline(13.7 mM NaCl, 1 mM phosphate, 0.27 mM KCl, pH 7.4), containing 10%trifluoroethanol, with the final peptide concentration of 50-100 μM. Theconcentrations of unfolded peptides were determined by the UV absorptionof the tyrosine residue at 276 nm in 6.0 M guanidinium hydrochlorideaqueous solution. The helix content of each peptide was determined fromthe mean residue CD at 222 nm, [θ]₂₂₂ (deg cm² dmol⁻¹) corrected for thenumber of amino acids. Percent helicity was calculated from the ratio[θ]₂₂₂/[θ]_(max), where [θ]_(max)=(−44000+250T)(1−k/n), with k=4.0 andn=number of residues. For details on θ_(max) calculations for HBShelices, see Wang et al., “Evaluation of Biologically Relevant Shortα-Helices Stabilized by a Main-chain Hydrogen-bond Surrogate,” J. Am.Chem. Soc. 128:9248-56 (2006), which is hereby incorporated by referencein its entirety.

Example 3 Affinity of Peptides for Ras Protein as Determined byFluorescence Polarization

The relative affinity of peptides for N-terminal His₆-tagged Ras₁₋₁₆₆was determined using fluorescence polarization based binding assay withfluorescein labeled SOS peptides 7^(uncon)-Flu, HBS 7-Flu and HBS7^(mut)-Flu. The polarization experiments were performed with a DTX 880Multimode Detector (Beckman) at 25° C., with excitation and emissionwavelengths at 485 and 525 nm, respectively. All samples were preparedin 96 well plates in 0.1% pluronic F-68 (Sigma). Addition of anincreasing concentration (0 nm to 750 μM) of Ras₁₋₁₆₆ protein to a 15 nMsolution of fluorescein labeled SOS peptide in Ras₁₋₁₆₆ dialysis bufferafforded the saturation binding curve. The IC₅₀ value obtained from thisbinding curve was fit into equation (1) to calculate the dissociationconstant (K_(D)) for the Sos/Ras₁₋₁₆₆ complex. The binding affinity(K_(D)) values reported for each peptide are the averages of 3individual experiments, and were determined by fitting the experimentaldata to a sigmoidal dose-response nonlinear regression model on GraphPadPrism 4.0. Results are shown in FIG. 10.K _(D1)=(R _(T)*(1−F _(SB))+L _(ST) *F _(SB) ²)/F _(SB) −L _(ST)   (1)where:

-   R_(T)=Total concentration of Ras₁₋₁₆₆ protein-   L_(ST)=Total concentration of Sos fluorescent peptide-   F_(SB)=Fraction of bound Sos fluorescent peptide

Example 4 Assay for Inhibition of Sos-mediated Guanine NucleotideExchange Activity by Peptides of the Invention

Nucleotide exchange assays using mantGDP were performed as described byAhmadian et al., 2002; and Margarit et al., 2003. Briefly, purified Ras(residues 1-166 of human Ha-Ras) was incubated in an equimolar amount ofmantGDP in the presence of 4 mM EDTA in exchange buffer (20 mM Tris [pH7.4], 50 mM NaCl). Reactions were stopped with 14 mM MgCl₂. Nucleotidedissociation rates were measured by incubation of 1 μM Ras•mantGDP inreaction buffer (20 mM Tris [pH 7.4], 14 mM MgCl2, and 50 mM NaCl)supplemented with 25 μM peptides, 5 μM Sos-Cat and 100 μM unlabeled GDP.The data were fitted to a single exponential decay function using theprogram Prism (GraphPad Software Inc.). Results are shown in FIG. 4.

Example 5 GST Assays for Inhibition of Ras/Sos by Peptides of theInvention

GST-Ras fusion protein (1 μM), His-tagged Sos protein (1 μM) and theindicated amount of HBS 7 were added to 1 ml of binding buffer (20 mMTris (pH 7.6), 50 mM NaCl, 1 mM dithiothreitol, 5 mM EDTA, and 1% TritonX-100) and incubated at 4° C. for 30 min. Following incubation, 60 μl1:1of 1:1 slurry of glutathione-Sepharose 4B beads resuspended inbinding buffer were added to each sample. Samples were incubated for anadditional 20 min at 4° C. Beads were subsequently pelleted, washed fivetimes with binding buffer, and resuspended in SDS-polyacrylamide gelelectrophoresis sample buffer. Proteins were separated bySDS-polyacrylamide gel electrophoresis and transferred tonitrocellulose. Western blots were probed with anti-His antibody andanti-GST antibody to detect Sos and Ras, respectively, and the resultsare shown in FIG. 6.

Example 6 Ras Activation Assays

The RBD-pull down assay was carried out as described in Boykevisch S,Zhao C, Sondermann H, Philippidou P, Halegoua S, Kuriyan J, Bar-Sagi D.Regulation of ras signaling dynamics by Sos-mediated positive feedbackCurr Biol. 2006 Nov. 7; 16(21):2173-9 and as described herein.GST-Raf-RBD fusion proteins were expressed in E. coli by induction with0.5 mM of isopropyl-1-thio-β-D-galactopyranoside (IPTG) for 5 hours. Theexpressed fusion proteins were isolated from bacteria lysates byincubation with glutathione agarose beads for 1 hour at 4° C. HeLa cellswere grown to confluence, serum-starved for 4 hours, and incubated with75 μM peptide for an additional 12 hours. For experiments withSosCat-CAAX (SosCat with CAAX box of HRas), HeLa cells were transfectedwith HA-tagged SosCAAX twenty four hours prior to starvation. Cells weretreated with the indicated peptides for 12 hours prior to stimulation.After stimulation with 10 ng/ml EGF for the indicated intervals at 37°C., the cells were lysed in RBD lysis buffer containing 25 mM Tris-HCl(pH 7.4), 120 mM NaCl, 10 mM MgCl2, 1 mM EDTA, 10% glycerol, 10 mg,/mlpepstatin, 50 mM NaF, 1% aprotinin, 10 mg/ml leupeptin, 1 mM Na3VO4, 10mM benzamidine, 10 mg/ml soybean trypsin inhibitor, 1% NP40, and 0.25%sodium deoxycholic acid. The lysates were then incubated with 20 μg ofrecombinant GST-Raf-RBD immobilized to agarose beads for 1.5 hours at 4°C. The complexes were collected by centrifugation and washed six timeswith the RBD lysis buffer. Bound proteins were eluted with SDS samplebuffer, separated by SDS-12.5% PAGE and transferred to nitrocellulosemembrane. The proteins were detected by blotting with anti-HA (12CA5;1:10,000) for SosCatCAAX or anti-Ras10 (Millipore; 1:10,000) primaryantibodies and Alexa Fluor 680 goat anti-mouse (Molecular Probes,1:10,000) secondary antibody and visualized with the Odyssey InfraredImaging System (LiCor). Results are shown in FIG. 8.

Example 7 EGFR and Erk Activation Assays

Mitogen-activated protein kinase (MAPK) signal transduction pathways arewidespread mechanisms of eukaryotic cell regulation (FIG. 6). MAPK isinvolved in control of activities including cellular metabolism,motility, survival, apoptosis, and differentiation. Ras activation bySos is closely tied to the initiation of this signaling pathway, whichultimately leads to expression of a variety of genes including thosecontrolled by the serum response element within the IEG (immediatelyearly gene) promoter. Peptides were tested in an assay to determinetheir ability to inhibit Erk and/or EGFR activation. Cells were treatedand lysed as described above, and as described in Boykevisch S, Zhao C,Sondermann H, Philippidou P, Halegoua S, Kuriyan J, Bar-Sagi D.Regulation of ras signaling dynamics by Sos-mediated positive feedbackCurr Biol. 2006 Nov. 7; 16(21):2173-9, and Xu L, Lubkov V, Taylor L J,Bar-Sagi D. Feedback regulation of Ras signaling by Rabex-5-mediatedubiquitination. Curr Biol. 2010 Aug. 10; 20(15):1372-7, which are herebyincorporated by reference in their entirety. Levels of total ERK2 andphosphorylated ERK were detected with anti-ERK2 (Upstate Biotechnology,1:1,000) and phospho-ERK1/2 (Cell Signaling, 1:1,000) antibodies,respectively. ERK phosphorylation levels were quantified with theOdyssey software and normalized to total ERK expression. EGFR and pEGFRlevels were detected by blotting with anti-EGFR (Santa Cruz Biotech) andpEGFR pY1068 (Cell Signaling) antibodies. Results are shown in FIG. 8.

Example 8 Cellular Uptake Assays

HeLa cells were plated at sub-confluency in DMEM supplemented with 10%FBS in a 96 well plate with glass bottom. The following day, media wasreplaced with one supplemented with 1 μM fluorescein (5-FAM) only orfluorescein-tagged peptides as indicated. After 12 hours, the cells werewashed twice with warm PBS and imaged directly with the Zeiss Axiovert200M microscope.

Example 9 NMR Experiments

His6-Ras (1-166) and Sos-Cat (564-1049) Expression. His6-tagged HRas(residues 1-166) and His6-tagged SosCat (residues 550-1050) both inpProEx HTb expression vectors, were expressed in Escherichia coli (BL21)by induction with 500 μM IPTG at a cell density corresponding to anabsorbance of OD600=1.0. Pellets were resuspended in buffer containing20 mM Tris pH7.6, 200 mM NaCl, 2.5 mM MgCl2, 2 μMphenylmethylsulfonylfluride (PMSF), 1% aprotinin, 10 μg/ml leupeptin, 10mM benzamidine, 10 μg/ml soybean trypsin inhibitor, and 10 μg/mlpepstatin, and sonicated using a Branson Cell Disrupter 200. Clarifiedlysates containing polyhistidine tagged proteins were incubated withcharged nickel resin (Invitrogen) at 4° C. for 1 hour. The resin waswashed five times in resuspension buffer containing 50 mM imidazole. Thetagged proteins were eluded with buffer containing 200 mM imidazole in20 mM Tris pH 7.6, 200 mM NaCl. Eluted proteins were dialyzed againstbuffer containing 20 mM Tris pH 7.6 and 200 mM NaCl for His-taggedSosCat, and 20 mM Tris pH 7.6, 200 mM NaCl and 1 mM MgCl2 for His-taggedRas. The eluted proteins were concentrated with 5,000 kD molecularcut-off Amicon ultra centrifugal columns (Millipore). Purified proteinswere snap frozen in liquid N2 and stored at −80° C. till further use.

For ¹H—¹⁵N HSQC NMR experiments, BL21 cells harbouring the His-Rasconstruct were grown at 37° C. in M9 media supplemented with ¹⁵NH₄Cl asthe sole source of nitrogen¹⁵. Protein production was induced with 500μM IPTG at O.D. 1.0 for 16 hours at 16° C. Protein purification andconcentration were performed as in Section 5. The His₆-tag was removedby incubating the His6-tagged Ras with recombinant His6-tagged TobaccoEtch Virus (TEV) protease (Invitrogen) overnight at 4° C. followingmanufacturer's protocol. The sample was loaded on a charged NiNTAagarose column and the tag-less protein collected in the flow throughfraction. Uniformly ¹⁵N-labelled Ras was buffer exchanged against theNMR buffer (20 mM Na2HPO4-NaH2PO4, pH 5.5, 150 mM NaCl, 10 mM MgCl2)using Amicon Ultra centrifugal filter (Millipore) and supplemented with10% D₂O. Data was collected on a 900 MHz Bruker four-channel NMR systemequipped with cryoprobe at 30° C. and analyzed with the BioSpin software(Bruker). Mean chemical shift difference (ΔδNH) observed for ¹H and ¹⁵Nnuclei of various resonances corresponding to residues in the switch andnon-switch regions were calculated. Results of an NMR experiments areshown in FIG. 5.

Example 10 Peptide Design

Design of Ras/Sos inhibitors was performed starting with the wild-typeSos₉₂₉₋₉₄₄ α-H sequence. Additional modifications were introduced inorder to improve the solubility of the HBS peptides. Charged residueswere introduced at positions not involved in Ras binding. Computationalalanine scanning was performed on two separate crystal structures (PDBcodes: 1NVW and 1BKD) of the Ras/Sos complex to determine otherimportant binding residues in the α-H helix that may be incorporated inthe peptide mimetics. Additionally, non-essential β-branched residues(including threonine) in the wild-type α-H sequence were replaced withsuitable residues to afford peptides with higher helical content (Table1), as it was hypothesized that such modifications would improveα-helicity.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

What is claimed is:
 1. A peptide having a stable, internally-constrainedalpha-helix, wherein said alpha helix is constrained by a crosslinkformed by a carbon-carbon bond-forming reaction, and further wherein thepeptide mimics at least an alpha-helical portion of a protein capable ofinteracting with a Ras protein, and wherein the peptide comprises theformula:

where

is a single or double carbon-carbon bond;

is a single bond and is cis or trans when

is a double bond; n is 1 or 2; m is zero or any positive integer; R₁,R₂, R₃ and R₄ are independently hydrogen, an amino acid side chain, analkyl group, or an aryl group.
 2. The peptide of claim 1, wherein theprotein capable of interacting with a Ras protein is Sos.
 3. The peptideof claim 2, wherein the peptide mimics at least a portion of an αA, αB,αC, αD, αE, αF, αG, αH, αI, αJ, or αK helix of Sos.
 4. The peptide ofclaim 3, wherein the peptide mimics at least a portion of the α-H helixof Sos.
 5. The peptide of claim 4, wherein the peptide mimics aminoacids 929-944 of the α-H sequence of Sos.
 6. The peptide of claim 1,wherein the peptide comprises a sequence of the formula FXGZZXZXZLXZEXXN(SEQ ID NO: 1) where X is any amino acid residue and Z is an hydrophobicamino acid residue.
 7. The peptide of claim 1, wherein the peptidecomprises an amino acid sequence of Table 1, and has aninternally-constrained alpha-helix spanning residues 1 through 4 of theamino acid sequence.
 8. The peptide of claim 1, wherein m is
 1. 9. Thepeptide of claim 1, wherein m is
 2. 10. The peptide of claim 1, whereinn is
 1. 11. The peptide of claim 1, wherein n is
 2. 12. A pharmaceuticalcomposition comprising a peptide according to claim 1 and apharmaceutically acceptable vehicle.
 13. A method of inhibiting Rassignaling in a cell, comprising contacting the cell with an effectiveamount of a composition comprising a peptide according to claim
 1. 14.The method of claim 13, wherein the protein capable of interacting witha Ras protein is Sos.
 15. The method of claim 14, wherein the peptidemimics at least a portion of an αA, αB, αC, αD, αE, αF, αG, αH, αI, αJ,or αK helix of Sos.
 16. The method of claim 15, wherein the peptidemimics at least a portion of the α-H helix of Sos.
 17. The method ofclaim 16, wherein the peptide mimics amino acids 929-944 of the α-Hsequence of Sos.
 18. The method of claim 13, wherein the peptidecomprises a sequence of the formula FXGZZXZXZLXZEXXN (SEQ ID NO: 1)where X is any amino acid residue and Z is an hydrophobic amino acidresidue.
 19. The method of claim 13, wherein the peptide comprises anamino acid sequence of Table 1, and has an internally-constrainedalpha-helix spanning residues 1 through 4 of the amino acid sequence.20. The method of claim 13, wherein m is
 1. 21. The method of claim 13,wherein m is
 2. 22. The method of claim 13, wherein n is
 1. 23. Themethod of claim 13, wherein n is
 2. 24. A method of treating cancer in asubject in need thereof, comprising administering to the subject apeptide according to claim
 1. 25. The method of claim 24, wherein thepeptide comprises a sequence of the formula FXGZZXZXZLXZEXXN (SEQ IDNO: 1) where X is any amino acid residue and Z is an hydrophobic aminoacid residue.
 26. The method of claim 24, wherein the peptide comprisesan amino acid sequence of Table 1, and has an internally-constrainedalpha-helix spanning residues 1 through 4 of the amino acid sequence.27. The peptide of claim 1, wherein the peptide does not comprise thesequence FEG1YRLELLKAEEAN (SEQ ID NO: 9) or FEAIYRLELLKAEEAN (SEQ ID NO:16).